Invading pathogens are recognized by pattern recognition receptors (PRRs) of the innate immune system as a first line of defense. Activation of PRRs from the Nucleotide-binding and leucine-rich repeat containing (NLR) protein family and Absent in melanoma 2 (AIM2) in macrophages results in inflammasome formation. Inflammasomes are protein platforms that are essential for the activation of inflammatory Caspases and subsequently for the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1? and IL-18. Assembly of inflammasomes depends on PYRIN domain (PYD)-mediated recruitment of the adaptor protein ASC to activated PRRs, which then recruits pro-Caspase-1. In addition, Caspase-4, -5 and -8 participate. However, the molecular mechanism of inflammasome activation by cytosolic PRRs and in particular, regulation and termination of this process is poorly understood. We discovered a family of small proteins composed of only a PYD, called PYD-containing (PYDC or POP) proteins. We show that PYDCs function to inhibit inflammasome activation by blocking the PYD-PYD interactions essential for inflammasome formation. However, their precise role and contribution to in vivo host defense and inflammatory disease is still unknown. Inflammasomes are essential for host defense; however, inappropriate inflammasome activation also causes excessive and chronic inflammation, tissue destruction and the debilitating symptoms of the growing autoinflammatory diseases. Therefore, a balanced inflammasome response to allow pathogen clearance during acute infection, while preventing systemic inflammation through timely termination, is essential for homeostasis. We hypothesize that it is the role of the PYDC proteins to maintain this balanced inflammasome response through a negative feedback mechanism. Since PYDC proteins are lacking from mice, we generated novel mouse models to simulate the more complex inflammasome regulation evolved in humans. We propose to determine PYDC-mediated inflammasome regulation and termination during all known scenarios of inflammasome activation: pathogen infection/pathogen-associated molecular pattern (PAMP), tissue damage/damage associated molecular pattern (DAMP) and hereditary PRR mutation in macrophages and in vivo. We will focus on select inflammasome-activating PRRs. In aim 1 we will determine the molecular mechanism of inflammasome regulation and termination for each PYDC ex vivo in macrophages and in aim 2 we propose to determine PYDC-mediated inflammasome regulation in vivo. Collectively, our study is designed to establish the specific inflammasome regulatory function of the PYDC family during infection and inflammatory disease, which we expect will significantly advance our understanding of fundamental biological principles underlying innate immunity, host defense and inflammatory disease and the mechanism by which these responses are regulated.